The overriding hypothesis to be tested in this proposal is that liver-targeted delivery of adeno-associated virus (AAV) vectors can safely mediate long-term expression of therapeutic levels of coagulation factor VIII (FVIII) for the treatment of hemophilia A. We have already used this approach to safely generate stable, therapeutic levels of coagulation factor IX (FIX) in mice and non-human primates. In addition, we have recently initiated a phase I/II clinical trial of AAV-mediated, liver-targeted gene transfer for hemophilia B in which we have established stable, therapeutic FIX expression with a single vector dose in our first subject. Much of the pre- clinical data used to support this trial were generated under our current R01 grant. In this renewal application, we will expand our efforts to include hemophilia A, the most common inherited bleeding disorder. However, there are several unique challenges to gene transfer for hemophilia A. These include: 1) the large size of the gene encoding FVIII, 2) the poor efficiency of FVIII synthesis and secretion and 3) the high incidence of FVIII inhibitors. In addition, there is a continued need to improve the efficiency of both AAV production and AAV- mediated transgene expression. Based in these gaps in our knowledge we are proposing three Specific Aims in our renewal proposal. Aim 1: To assess the safety and efficacy of an AAV serotype 8 vector encoding a novel, potent FVIII-variant. Aim 2: To eradicate antibodies against FVIII and establish permanent tolerance following AAV-mediated, liver- targeted FVIII gene transfer. Aim 3: To improve the efficiency of AAV-mediated FVIII expression by preventing transcriptional silencing of vector genomes within transduced hepatocytes. We will perform these planned studies in our non-human primate model with vector produced in our GMP facility, thereby enabling careful evaluation in a highly relevant manner. We anticipate opening at least one clinical FVIII gene transfer trial based on the preclinical results generated from the experiments proposed in this renewal application. In addition, results from these studies will be of great utility not only for hemophilia A gene therapy but also other disorders that are potentially amenable to AAV-mediated, liver-targeted gene transfer. PUBLIC HEALTH RELEVANCE: The inadequacies of current therapy for hemophilia A have fuelled interest in alternative treatment approaches, including gene transfer. Successful AAV-mediated FVIII gene transfer for hemophilia A offers the potential of effective lifetime prevention of bleeding and its complications for patients with this disorder. In addition, the information gained from this study will be important in planning additional future strategies with AAV vectors in which other disorders affecting the liver, such as lysosomal storage and urea cycle disorders, are targeted.